Autoantigen-like protein

ABSTRACT

? The invention provides an autoantigen-like protein (AUTOP) and polynucleotides which identify and encode AUTOP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of AUTOP.

[0001] This application is a divisional application of U.S. applicationSer. No. 08/928,442, filed Sep. 12, 1997, all of which applications andpatents are hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a human autoantigen-like protein and to the use of these sequences inthe diagnosis, prevention, and treatment of immune disorders.

BACKGROUND OF THE INVENTION

[0003] Autoimmune disorders occur when an immune response is directed toendogenous or “self” proteins that are recognized as “foreign” antigens.Immune response consists of the production of antibodies and theactivation of T cells that react with these antigens and initiate aseries of destructive changes. The tolerance to “self” antigens isinduced in early lymphocyte development by the specific clonal deletionof cells which recognize “self” antigens and is supported by cell-cellinteractions, cytokine and immunosuppressive factors, and induction ofanergy. The breakdown of tolerance to “self” proteins can occur by avariety of mechanisms. T cells can be stimulated by cross-reactingmicrobial antigens which have features in common with self antigens, asin rheumatic fever and chronic synovial inflammatory processes. Normallysequestered proteins that become exposed to immune surveillance throughtissue changes, as in Hashimoto's thyroiditis, will activate apopulation of cells that were not originally clonally deleted. There isa correlation between specific major histocompatibility loci (MHC) andsusceptibility to certain autoimmune disorders, indicating that multiplecomponents may contribute to disease induction.

[0004] Insulin-dependent diabetes mellitus (IDDM) is an autoimmunedisease that results from the destruction of the insulin-secretingbeta-cells of the pancreas. Patients with IDDM have insulitis, alymphocytic infiltration of the islets of Langerhans, islet-specific Th1lymphocytes, and antibodies directed against components of the isletcells. IDDM in animal models is T cell mediated and requires theparticipation of both CD8+, class I MHC restricted and CD4+, class IIMHC restricted T cells. There is a demonstrated association between MHCclass II DR4 polymorphic alleles and disease susceptibility, indicatingthat the response is antigen driven. Peptide elution studies indicatethat these polymorphisms may modify suceptability to IDDM by selectivelyaffecting the nature of the molecules presented to T cells by theseclass II molecules (Wicker, L. S. et al. (1995) Annu. Rev. Immunol. 13:179-200; Reich, E. P. et al. (1994) J. Immunol. 152: 2279-2288).

[0005] Several beta-cell proteins have been identified as candidateantigens in IDDM. Antibodies to two glutamate acid decarboxylaseisoforms, insulin, carboxypeptidase H, ICA 516 and 64 kD integralmembrane proteins, hsp65, and several secretory granule protein havebeen found in the sera of diabetic and prediabetic individuals.Peripheral blood T cells from a majority of persons newly diagnosed withIDDM respond to a variety of insulin-secretory granule antigens. A mouseinsulin-secretory granule antigen (imogen 38) has recently been isolatedby using an assay based on the proliferative response of a human IDDM Tcell clone. Although this isolated antigen is a mitochondrial proteinwhich occurs in various tissues the imogen 38-reactive antibodies arefound only in the diseased pancreatic tissue (Roep, B. O. (1996)Diabetes 45: 1147-1156; and Arden, S. A. et al (1996) J. Clin. Invest.97: 551-561).

[0006] The discovery of an autoantigen-like protein and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention and treatmentof immune disorders.

SUMMARY OF THE INVENTION

[0007] The invention features a substantially purified polypeptide,autoantigen-like protein (AUTOP), having the amino acid sequence shownin SEQ ID NO: 1, or fragments thereof.

[0008] The invention further provides an isolated and substantiallypurified polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or fragments thereof and acomposition comprising said polynucleotide sequence. The invention alsoprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence encoding the amino acidsequence SEQ ID NO: 1, or fragments of said polynucleotide sequence. Theinvention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO: 1, or fragments or variants of saidpolynucleotide sequence.

[0009] The invention also provides an isolated and purified sequencecomprising SEQ ID NO: 2 or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO: 2. The inventionalso provides a polynucleotide sequence comprising the complement of SEQID NO: 2, or fragments or variants thereof.

[0010] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0011] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding AUTOP under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

[0012] The invention also provides a pharmaceutical compositioncomprising a substantially purified AUTOP having the amino acid sequenceof SEQ ID NO: 1 in conjunction with a suitable pharmaceutical carrier.

[0013] The invention also provides a purified antagonist of thepolypeptide of SEQ ID NO: 1. In one aspect the invention provides apurified antibody which binds to a polypeptide comprising the amino acidsequence of SEQ ID NO: 1.

[0014] Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO: 1.

[0015] The invention also provides a method for treating or preventingan immune disorder comprising administering to a subject in need of suchtreatment an effective amount of an antagonist to AUTOP.

[0016] The invention also provides a method for detecting apolynucleotide which encodes AUTOP in a biological sample comprising thesteps of: a) hybridizing the complement of the polynucleotide sequencewhich encodes SEQ ID NO: 1 to nucleic acid material of a biologicalsample, thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding AUTOP in the biologicalsample. In one aspect the nucleic acid material of the biological sampleis amplified by the polymerase chain reaction prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIGS. 1A, 1B, 1C and 1D show the amino acid sequence (SEQ IDNO: 1) and nucleic acid sequence (SEQ ID NO: 2) of AUTOP. The alignmentwas produced using MacDNASIS PRO™ software (Hitachi Software EngineeringCo. Ltd. San Bruno, Calif.).

[0018]FIGS. 2A and 2B show the amino acid sequence alignments betweenAUTOP (SEQ ID NO: 1) and mouse imogen 38 (GI 1272669; SEQ ID NO: 3),produced using the multisequence alignment program of DNASTAR software(DNASTAR Inc, Madison Wis.).

DESCRIPTION OF THE INVENTION

[0019] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0020] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0021] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0022] Definitions

[0023] AUTOP, as used herein, refers to the amino acid sequences ofsubstantially purified AUTOP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0024] The term “agonist”, as used herein, refers to a molecule which,when bound to AUTOP, increases or prolongs the duration of the effect ofAUTOP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of AUTOP.

[0025] An “allele” or “allelic sequence”, as used herein, is analternative form of the gene encoding AUTOP. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0026] “Altered” nucleic acid sequences encoding AUTOP as used hereininclude those with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent AUTOP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding AUTOP,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding AUTOP. The encoded protein may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent AUTOP.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of AUTOP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

[0027] “Amino acid sequence” as used herein refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of AUTOP arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of AUTOP. Where “aminoacid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

[0028] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0029] The term “antagonist” as used herein, refers to a molecule which,when bound to AUTOP, decreases the amount or the duration of the effectof the biological or immunological activity of AUTOP. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of AUTOP.

[0030] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind AUTOPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0031] The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0032] The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

[0033] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic AUTOP, orany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0034] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

[0035] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding AUTOP (SEQ ID NO: 1) or fragments thereof (e.g., SEQ ID NO: 2and fragments thereof) may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

[0036] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, has been extendedusing XL-PCR (Applied Biosystems, Foster City Calif.) in the 5′ and/orthe 3′ direction and resequenced, or has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly (e.g., GELVIEW™ Fragment Assembly system,GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

[0037] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO: 2 by northern analysis is indicativeof the presence of mRNA encoding AUTOP in a sample and therebycorrelates with expression of the transcript from the polynucleotideencoding the protein.

[0038] A “deletion”, as used herein, refers to a change in the aminoacid or nucleotide sequence and results in the absence of one or moreamino acid residues or nucleotides.

[0039] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to AUTOP or theencoded AUTOP. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

[0040] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

[0041] Human artificial chromosomes (HACs) are linear microchromosomeswhich may contain DNA sequences of 10K to 10M in size and contain all ofthe elements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

[0042] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0043] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0044] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0045] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0046] “Microarray” refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

[0047] The term “modulate”, as used herein, refers to a change in theactivity of AUTOP. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional or immunological properties of AUTOP.

[0048] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments thereof,and to DNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand.“Fragments” are those nucleic acid sequences which are greater than 60nucleotides than in length, and most preferably includes fragments thatare at least 100 nucleotides or at least 1000 nucleotides, and at least10,000 nucleotides in length.

[0049] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to about 60 nucleotides, preferably about15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides,which can be used in PCR amplification or a hybridization assay, or amicroarray. As used herein, oligonucleotide is substantially equivalentto the terms “amplimers”, “primers”, “oligomers”, and “probes”, ascommonly defined in the art.

[0050] “Peptide nucleic acid”, PNA as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least five nucleotides in length linked to a peptide backbone ofamino acid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

[0051] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from five amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ ID NO:1” encompasses the full-length AUTOP and fragments thereof.

[0052] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding AUTOP,or fragments thereof, or AUTOP itself may comprise a bodily fluid,extract from a cell, chromosome, organelle, or membrane isolated from acell, a cell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport, a tissue, a tissue print, and the like.

[0053] The terms “specific binding” or “specifically binding”, as usedherein, refers to that interaction between a protein or peptide and anagonist, an antibody and an antagonist. The interaction is dependentupon the presence of a particular structure (i.e., the antigenicdeterminant or epitope) of the protein recognized by the bindingmolecule. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabeled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

[0054] The terms “stringent conditions” or “stringency”, as used herein,refer to the conditions for hybridization as defined by the nucleicacid, salt, and temperature. These conditions are well known in the artand may be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

[0055] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0056] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0057] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0058] A “variant” of AUTOP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0059] The Invention

[0060] The invention is based on the discovery of a new humanautoantigen-like protein (hereinafter referred to as “AUTOP”), thepolynucleotides encoding AUTOP, and the use of these compositions forthe diagnosis, prevention, or treatment of immune disorders.

[0061] Nucleic acids encoding the AUTOP of the present invention werefirst identified in Incyte Clone 035842 from the HUV-EC-C endothelialcell line cDNA library (HUVENOB01) using a computer search for aminoacid sequence alignments. A consensus sequence, SEQ ID NO: 2, wasderived from the following overlapping and/or extended nucleic acidsequences: Incyte Clones 035842 (HUVENOB01), 2376448 (ISLTNOT01),1359659 (LUNGNOT12), 1649243 (PROSTUT09), and 437847 (THYRNOT01).

[0062] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1, as shown in FIGS.1A, 1B, 1C, and 1D. AUTOP is 395 amino acids in length and has apotential cAMP-dependent phosphorylation site at residue T₁₄₀, and eightpotential casein kinase II phosphorylation sites at residues at residuesS₆₈, T₈₀, S₉₀, T₉₅, T173, S₂₂₀, S₂₇₆, and T₂₈₈. In addition, AUTOP has apotential tyrosine kinase phosphorylation site at residue T₂₂₅, andtwelve potential protein kinase II phosphorylation sites at residuesT₃₅, T₄₀, S₆₈, S₇₄, T₈₀, S₉₀, T₉₅, T₁₁₇, T₁₇₂, T₂₁₅, T₂₅₂, and S₃₆₅. Asshown in FIGS. 2A and 2B, AUTOP has chemical and structural homologywith mouse imogen 38 (GI 1272669; SEQ ID NO: 3). In particular, AUTOPand mouse imogen 44 share 65% identity. Northern analysis shows theexpression of this sequence in various libraries, at least 63% of whichshow increased cell proliferation associated with cancer or immuneresponse.

[0063] The invention also encompasses AUTOP variants. A preferred AUTOPvariant is one having at least 80%, and more preferably at least 90%,amino acid sequence identity to the AUTOP amino acid sequence (SEQ IDNO: 1) and which retains at least one biological, immunological or otherfunctional characteristic or activity of AUTOP. A most preferred AUTOPvariant is one having at least 95% amino acid sequence identity to SEQID NO: 1.

[0064] The invention also encompasses polynucleotides which encodeAUTOP. Accordingly, any nucleic acid sequence which encodes the aminoacid sequence of AUTOP can be used to produce recombinant moleculeswhich express AUTOP. In a particular embodiment, the inventionencompasses the polynucleotide comprising the nucleic acid sequence ofSEQ ID NO: 2 as shown in FIGS. 1A, 1B, 1C, and 1D.

[0065] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding AUTOP, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring AUTOP, and all suchvariations are to be considered as being specifically disclosed.

[0066] Although nucleotide sequences which encode AUTOP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring AUTOP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding AUTOP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding AUTOP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0067] The invention also encompasses production of DNA sequences, orfragments thereof, which encode AUTOP and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding AUTOP or any fragment thereof.

[0068] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO: 2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

[0069] Methods for DNA sequencing which are well known and generallyavailable in the art and may be used to practice any of the embodimentsof the invention. These methods employ enzymes such as the Klenowfragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase andthermostable T7 DNA polymerase (Amersham Pharmacia Biotech (APB),Piscataway N.J.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(Life Technologies, Gaithersburg Md.). Preferably, sequence preparationis automated with machines such as the MICROLAB 2200 system (Hamilton,Reno Nev.) and the DNA ENGINE thermal cycler (MJ Research, WatertownMass.). Machines commonly used for sequencing include the ABI PRISM3700, 377 or 373 DNA sequencing systems (Applied Biosystems), theMEGABACE 1000 DNA sequencing system (APB), and the like.

[0070] The nucleic acid sequences encoding AUTOP may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0071] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0072] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

[0073] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. Also, random-primed libraries are preferable, in that theywill contain more sequences which contain the 5′ regions of genes. Useof a randomly primed library may be especially preferable for situationsin which an oligo d(T) library does not yield a full-length cDNA.Genomic libraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0074] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Applied Biosystems) and the entire process fromloading of samples to computer analysis and electronic data display maybe computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA which might bepresent in limited amounts in a particular sample.

[0075] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode AUTOP may be used in recombinant DNAmolecules to direct expression of AUTOP, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressAUTOP.

[0076] As will be understood by those of skill in the art, it may beadvantageous to produce AUTOP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0077] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterAUTOP encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0078] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding AUTOP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of AUTOP activity, it may be useful toencode a chimeric AUTOP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the AUTOP encoding sequence and theheterologous protein sequence, so that AUTOP may be cleaved and purifiedaway from the heterologous moiety.

[0079] In another embodiment, sequences encoding AUTOP may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Symp. Ser.7:215-223, Horn, T. et al. (1980) Nucl. Acids Symp. Ser. 7:225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of AUTOP, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A peptide synthesizer (Applied Biosystems).

[0080] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of AUTOP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0081] In order to express a biologically active AUTOP, the nucleotidesequences encoding AUTOP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0082] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding AUTOPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0083] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding AUTOP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0084] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Life Technologies) and the like maybe used. The baculovirus polyhedrin promoter may be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO; and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) may be cloned intothe vector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding AUTOP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0085] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for AUTOP. For example, whenlarge quantities of AUTOP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding AUTOP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0086] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0087] In cases where plant expression vectors are used, the expressionof sequences encoding AUTOP may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0088] An insect system may also be used to express AUTOP. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding AUTOPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of AUTOP will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which AUTOP may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0089] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding AUTOP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing AUTOP in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0090] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

[0091] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding AUTOP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding AUTOP, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0092] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0093] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress AUTOP may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0094] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0095] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding AUTOP isinserted within a marker gene sequence, transformed cells containingsequences encoding AUTOP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding AUTOP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0096] Alternatively, host cells which contain the nucleic acid sequenceencoding AUTOP and express AUTOP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0097] The presence of polynucleotide sequences encoding AUTOP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding AUTOP.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding AUTOP todetect transformants containing DNA or RNA encoding AUTOP.

[0098] A variety of protocols for detecting and measuring the expressionof AUTOP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson AUTOP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0099] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding AUTOPinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding AUTOP, or any fragments thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland,Ohio). Suitable reporter molecules or labels, which may be used for easeof detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0100] Host cells transformed with nucleotide sequences encoding AUTOPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode AUTOP may be designed to contain signal sequences which directsecretion of AUTOP through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding AUTOP tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and AUTOP may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingAUTOP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying AUTOP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0101] In addition to recombinant production, fragments of AUTOP may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using an ABI 431A peptidesynthesizer (Applied Biosystems). Various fragments of AUTOP may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0102] Therapeutics

[0103] Based on the chemical and structural homology between AUTOP andthe imogen 38 antigen from mouse (GI 1272669), AUTOP appears to be anautoantigen involved in the β-cell destruction process. Autoantigentargets of an immune response can be primary recognition targets orsecondary targets generated as a result of the primary immune response.Therefore, AUTOP appears to play a role in immune disorders.

[0104] In one embodiment, an antagonist of AUTOP may be administered toa subject to prevent or treat an immune disorder. Such disorders mayinclude, but are not limited to, AIDS, Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis, andextracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections; and trauma. In one aspect, anantibody which specifically binds AUTOP may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express AUTOP.

[0105] In another embodiment, a vector expressing the complement of thepolynucleotide encoding AUTOP may be administered to a subject to treator prevent an immune disorder including, but not limited to, thosedescribed above.

[0106] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0107] An antagonist of AUTOP may be produced using methods which aregenerally known in the art. In particular, purified AUTOP may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind AUTOP.

[0108] Antibodies to AUTOP may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0109] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith AUTOP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0110] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to AUTOP have an amino acid sequenceconsisting of at least five amino acids and more preferably at least 10amino acids. It is also preferable that they are identical to a portionof the amino acid sequence of the natural protein, and they may containthe entire amino acid sequence of a small, naturally occurring molecule.Short stretches of AUTOP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0111] Monoclonal antibodies to AUTOP may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0112] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceAUTOP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0113] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0114] Antibody fragments which contain specific binding sites for AUTOPmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0115] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between AUTOP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering AUTOP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0116] In another embodiment of the invention, the polynucleotidesencoding AUTOP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding AUTOP may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding AUTOP. Thus, complementary molecules orfragments may be used to modulate AUTOP activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding AUTOP.

[0117] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encodingAUTOP. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

[0118] Genes encoding AUTOP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes AUTOP. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0119] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the geneencoding AUTOP (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0120] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding AUTOP.

[0121] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0122] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding AUTOP. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA constitutively or inducibly can be introduced into cell lines,cells, or tissues.

[0123] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0124] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposome injectionsor polycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

[0125] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0126] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of AUTOP,antibodies to AUTOP, mimetics, agonists, antagonists, or inhibitors ofAUTOP. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0127] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0128] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0129] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0130] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0131] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0132] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0133] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers may also be used for delivery.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0134] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0135] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0136] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic acids, etc. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0137] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of AUTOP, such labeling wouldinclude amount, frequency, and method of administration.

[0138] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0139] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0140] A therapeutically effective dose refers to that amount of activeingredient, for example AUTOP or fragments thereof, antibodies of AUTOP,agonists, antagonists or inhibitors of AUTOP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0141] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0142] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0143] Diagnostics

[0144] In another embodiment, antibodies which specifically bind AUTOPmay be used for the diagnosis of conditions or diseases characterized byexpression of AUTOP, or in assays to monitor patients being treated withAUTOP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for AUTOP includemethods which utilize the antibody and a label to detect AUTOP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0145] A variety of protocols including ELISA, RIA, and FACS formeasuring AUTOP are known in the art and provide a basis for diagnosingaltered or abnormal levels of AUTOP expression. Normal or standardvalues for AUTOP expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to AUTOP under conditions suitable for complex formation.The amount of standard complex formation may be quantified by variousmethods, but preferably by photometric means. Quantities of AUTOPexpressed in subject samples, control and diseased, from biopsiedtissues are compared with the standard values. Deviation betweenstandard and subject values establishes the parameters for diagnosingdisease.

[0146] In another embodiment of the invention, the polynucleotidesencoding AUTOP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof AUTOP may be correlated with disease. The diagnostic assay may beused to distinguish between absence, presence, and excess expression ofAUTOP, and to monitor regulation of AUTOP levels during therapeuticintervention.

[0147] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding AUTOP or closely related molecules, may be used to identifynucleic acid sequences which encode AUTOP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding AUTOP, alleles, or related sequences.

[0148] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the AUTOP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO: 2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring AUTOP.

[0149] Means for producing specific hybridization probes for DNAsencoding AUTOP include the cloning of nucleic acid sequences encodingAUTOP or AUTOP derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as 32P or 35S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0150] Polynucleotide sequences encoding AUTOP may be used for thediagnosis of conditions or disorders which are associated withexpression of AUTOP. Examples of such conditions or disorders includeAIDS, Addison's disease, adult respiratory distress syndrome, allergies,anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn'sdisease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, and extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections; and trauma. The polynucleotide sequences encoding AUTOP maybe used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered AUTOP expression. Such qualitative orquantitative methods are well known in the art.

[0151] In a particular aspect, the nucleotide sequences encoding AUTOPmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding AUTOP may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding AUTOP in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0152] In order to provide a basis for the diagnosis of diseaseassociated with expression of AUTOP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes AUTOP,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0153] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0154] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0155] Additional diagnostic uses for oligonucleotides designed from thesequences encoding AUTOP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5′→3′) and another with antisense (3′←5′),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0156] Methods which may also be used to quantitate the expression ofAUTOP include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P. C. et al.(1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.Biochem. 212:229-236). The speed of quantitation of multiple samples maybe accelerated by running the assay in an ELISA format where theoligomer of interest is presented in various dilutions and aspectrophotometric or colorimetric response gives rapid quantitation.

[0157] In further embodiments, an oligonucleotide derived from any ofthe polynucleotide sequences described herein may be used as a target ina microarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55).

[0158] In one embodiment, the microarray is prepared and used accordingto the methods described in PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

[0159] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5′, or 3′, sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

[0160] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

[0161] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0162] In order to conduct sample analysis using a microarray, the RNAor DNA from a biological sample is made into hybridization probes. ThemRNA is isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

[0163] In another embodiment of the invention, the nucleic acidsequences which encode AUTOP may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome or to artificial chromosomeconstructions, such as human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0164] Fluorescent in situ hybridization (FISH as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at Online MendelianInheritance in Man (OMIM). Correlation between the location of the geneencoding AUTOP on a physical chromosomal map and a specific disease , orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

[0165] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0166] In another embodiment of the invention, AUTOP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenAUTOP and the agent being tested, may be measured.

[0167] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to AUTOP largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with AUTOP, or fragments thereof, and washed.Bound AUTOP is then detected by methods well known in the art. PurifiedAUTOP can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0168] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding AUTOPspecifically compete with a test compound for binding AUTOP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with AUTOP.

[0169] In additional embodiments, the nucleotide sequences which encodeAUTOP may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0170] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0171] I HUVENOB01 cDNA Library Construction

[0172] The HUVEC cell line is a homogeneous, well characterized, earlypassage endothelial cell culture derived from human umbilical vein (CellSystems Corporation, Kirkland, Wash. 98034). The HUVENOB01 cDNA librarywas custom constructed by Stratagene. Poly(A+)RNA (mRNA) was purifiedfrom HUVEC cells. cDNA synthesis was primed either with oligo d(T) orrandom hexamers and the two cDNA copies were treated separately.Synthetic adaptor oligonucleotides were ligated onto cDNA ends enablingits insertion into UNI-ZAP vector system (Stratagene). Additionalunidirectional vectors include pcDNAI (Invitrogen) and pSHlox-1(Novagen). The two libraries were combined into a single library bymixing equal numbers of bacteriophage. These library phage particleswere used to infect E. coli host strain XL1-BLUE (Stratagene).

[0173] II Isolation and Sequencing of cDNA Clones

[0174] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process, in which the host bacterial strain wascoinfected with both the lambda library phage and an f1 helper phage.Polypeptides derived from both the library-containing phage and thehelper phage nicked the lambda DNA, initiated new DNA synthesis fromdefined sequences on the lambda target DNA and created a smaller, singlestranded circular phagemid DNA molecule that included all DNA sequencesof the PBLUESCRIPT plasmid and the cDNA insert. The phagemid DNA wassecreted from the cells and purified, then used to re-infect fresh hostcells, where the double stranded phagemid DNA was produced.

[0175] Phagemid DNA was purified using the Magic Minipreps DNAPurification System (Promega). Phagemid DNA was also purified using theQIAwell-8 Plasmid Purification System from QIAGEN, QiAwell PLUS andQIAwell ULTRA DNA Purification System (QIAGEN Inc., Chatsworth, Calif.).The DNA was eluted from the purification resin already prepared for DNAsequencing and other analytical manipulations.

[0176] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0177] The nucleotide sequences of the Sequence Listing or amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases which contain previously identified and annotated sequenceswere searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul S F (1993) J MolEvol 36:290-300; Altschul, S F et al (1990) J Mol Biol 215:403-410).

[0178] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R F and T F Smith (1992Protein Engineering 5:35-51), incorporated herein by reference, can beused when dealing with primary sequence patterns and secondary structuregap penalties. As disclosed in this application, the minimum length ofthe sequences in the Sequence Listing is 49 nucleotides, and the upperlimit of uncalled bases where N is recorded rather than A, C, G, or T is12%.

[0179] The BLAST approach, as detailed in Karlin and Altschul (1993;Proc Nat Acad Sci 90:5873-7) and incorporated herein by referencesearches matches between a query sequence and a database sequence, toevaluate the statistical significance of any matches found, and toreport only those matches which satisfy the user-selected threshold ofsignificance. In this application, threshold was set at 10-25 fornucleotides and 10-14 for peptides.

[0180] Incyte nucleotide sequence were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian sequences(mam), and deduced amino acid sequences from the same clones aresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology. The relevantdatabase for a particular match were reported as a Glxxx±p (where xxx ispri, rod, etc and if present, p= peptide) as shown in Table 1.

[0181] IV Northern Analysis

[0182] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0183] Analogous computer techniques using BLAST (Altschul, S. F. (1993)supra; Altschul, S. F. et al. (1990) supra) are used to search foridentical or related molecules in nucleotide databases such as GenBankor the LIFESEQ database (Incyte Genomics, Inc., Palo Alto, Calif.). Thisanalysis is much faster than multiple, membrane-based hybridizations. Inaddition, the sensitivity of the computer search can be modified todetermine whether any particular match is categorized as exact orhomologous.

[0184] The basis of the search is the product score which is defined as:

% sequence identity×% maximum BLAST score/100

[0185] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0186] The results of northern analysis are reported as a list oflibraries in which the transcript encoding AUTOP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0187] V Extension of AUTOP Encoding Polynucleotides

[0188] The nucleic acid sequence of the Incyte Clone 035842 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension in the antisense direction, and the other was synthesized toextend sequence in the sense direction. Primers were used to facilitatethe extension of the known sequence “outward” generating ampliconscontaining new, unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06(National Biosciences), or another appropriate program, to be about 22to about 30 nucleotides in length, to have a GC content of 50% or more,and to anneal to the target sequence at temperatures of about 68° toabout 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

[0189] Selected human cDNA libraries (Life Technologies) were used toextend the sequence If more than one extension is necessary or desired,additional sets of primers are designed to further extend the knownregion.

[0190] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Applied Biosystems) and thoroughlymixing the enzyme and reaction mix. Beginning with 40 pmol of eachprimer and the recommended concentrations of all other components of thekit, PCR was performed using the DNA ENGINE thermal cycler (M.J.Research) and the following parameters: Step 1 94° C. for 1 min (initialdenaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 min Step 4 94°C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 min Step 7Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 sec Step 965° C. for 1 min Step 10 68° C. for 7:15 mm Step 11 Repeat step 8-10 for12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0191] A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQUICK (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

[0192] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

[0193] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0194] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0195] In like manner, the nucleotide sequence of SEQ ID NO: 2 is usedto obtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0196] VI Labeling and Use of Individual Hybridization Probes

[0197] Hybridization probes derived from SEQ ID NO: 2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate (AmershamPharmacia Biotech) and T4 polynucleotide kinase (DuPont NEN, Boston,Mass.). The labeled oligonucleotides are substantially purified withSEPHADEX G-25 superfine resin column (Amersham Pharmacia Biotech). Aaliquot containing 10⁷ counts per minute of the labeled probe is used ina typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases (Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II; DuPont NEN).

[0198] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patternsare visualized using autoradiography or an alternative imaging means andcompared.

[0199] VII Microarrays

[0200] To produce oligonucleotides for a microarray, the nucleotidesequence described herein is examined using a computer algorithm whichstarts at the 3′ end of the nucleotide sequence. The algorithmidentifies oligomers of defined length that are unique to the gene, havea GC content within a range suitable for hybridization, and lackpredicted secondary structure that would interfere with hybridization.The algorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

[0201] In the alternative, a chemical coupling procedure and an ink jetdevice are used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

[0202] VIII Complementary Polynucleotides

[0203] Sequence complementary to the AUTOP-encoding sequence, or anypart thereof, is used to decrease or inhibit expression of naturallyoccurring AUTOP. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of AUTOP, SEQ ID NO: 1. To inhibit transcription, acomplementary oligonucleotide is designed from the most unique 5′sequence and used to prevent promoter binding to the coding sequence. Toinhibit translation, a complementary oligonucleotide is designed toprevent ribosomal binding to the AUTOP-encoding transcript.

[0204] IX Expression of AUTOP

[0205] Expression of AUTOP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express AUTOP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0206] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of AUTOP into the bacterial growth media which can be useddirectly in the following assay for activity.

[0207] X Demonstration of AUTOP Activity

[0208] AUTOP can be expressed by transforming a mammalian cell line suchas COS7, HeLa or CHO with an eukaryotic expression vector encodingAUTOP. Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. The cells are incubated for 48-72 hours after transformationunder conditions appropriate for the cell line to allow expression ofAUTOP.

[0209] The cell monolayer in the culture vessel is washed withphosphate-buffered saline to remove residual media, and the cells arelysed by addition of a buffer containing SDS using techniques well knownin the art. Portions of the cell lysate are heated in a boiling waterbath for five minutes and then applied to the wells of a polyacrylamidegel. The samples are electrophoresed at constant current usingtechniques well known in the art until a suitable tracking dye, such asbromophenol blue (Sigma, St. Louis, Mo.), has migrated to the bottom ofthe gel. Appropriate control samples, prepared from culture media andextracts of untransformed cells and/or cells transformed with vectorsequences alone, are electrophoresed in parallel lanes of the gel.Protein standards of known molecular weight (BioRad, Richmond, Calif.)are run in adjacent lanes to calibrate the gel.

[0210] The separated proteins are blotted onto a nitrocellulose membraneby techniques well known in the art. The presence of AUTOP is confirmedusing serum from recently diagnosed IDDM patients by the western blotprocedure that is well known in the art. The membrane is incubated withdilutions of serum from recently diagnosed IDDM patients. After washingto remove unbound antibodies, the membrane is incubated with tagged goatanti-human immunoglobulins. The tag may consist of any of a number ofchromogenic, fluorescent or enzymatic molecules that can be attached toimmunoglobulins by techniques well known in the art. The presence oftagged goat immunoglobulins bound to AUTOP can be detected usingtechniques appropriate to the nature of the tag. Sera from non IDDMpatients may be used as suitable controls for nonspecific binding to themembrane.

[0211] AUTOP will be detected by sera from recently diagnosed IDDMpatients, and not detected on blots incubated with sera from non IDDMpatients.

[0212] XI Production of AUTOP Specific Antibodies

[0213] AUTOP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO: 2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

[0214] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems peptide synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio iodinated, goat anti-rabbit IgG.

[0215] XII Purification of Naturally Occurring AUTOP Using SpecificAntibodies

[0216] Naturally occurring or recombinant AUTOP is substantiallypurified by immunoaffinity chromatography using antibodies specific forAUTOP. An immunoaffinity column is constructed by covalently couplingAUTOP antibody to an activated chromatographic resin, such asCNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, theresin is blocked and washed according to the manufacturer'sinstructions.

[0217] Media containing AUTOP is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of AUTOP (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/AUTOP binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and AUTOP iscollected.

[0218] XIII Identification of Molecules Which Interact with AUTOP

[0219] AUTOP or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled AUTOP, washed and any wells withlabeled AUTOP complex are assayed. Data obtained using differentconcentrations of AUTOP are used to calculate values for the number,affinity, and association of AUTOP with the candidate molecules.

[0220] The entire disclosure of all publications, patents andapplications mentioned herein in the above specification are herebyexpressly incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 3 395 amino acids amino acid single linear not provided HUVENOB0135842 1 Met Phe Pro Arg Val Ser Thr Phe Leu Pro Leu Arg Pro Leu Ser Arg1 5 10 15 His Pro Leu Ser Ser Gly Ser Pro Glu Thr Ser Ala Ala Ala IleMet 20 25 30 Leu Leu Thr Val Arg His Gly Thr Val Arg Tyr Arg Ser Ser AlaLeu 35 40 45 Leu Ala Arg Thr Lys Asn Asn Ile Gln Arg Tyr Phe Gly Thr AsnSer 50 55 60 Val Ile Cys Ser Lys Lys Asp Lys Gln Ser Val Arg Thr Glu GluThr 65 70 75 80 Ser Lys Glu Thr Ser Glu Ser Gln Asp Ser Glu Lys Glu AsnThr Lys 85 90 95 Lys Asp Leu Leu Gly Ile Ile Lys Gly Met Lys Val Glu LeuSer Thr 100 105 110 Val Asn Val Arg Thr Thr Lys Pro Pro Lys Arg Arg ProLeu Lys Ser 115 120 125 Leu Glu Ala Thr Leu Gly Arg Leu Arg Arg Ala ThrGlu Tyr Ala Pro 130 135 140 Lys Lys Arg Ile Glu Pro Leu Ser Pro Glu LeuVal Ala Ala Ala Ser 145 150 155 160 Ala Val Ala Asp Ser Leu Pro Phe AspLys Gln Thr Thr Lys Ser Glu 165 170 175 Leu Leu Ser Gln Leu Gln Gln HisGlu Glu Glu Ser Arg Ala Gln Arg 180 185 190 Asp Ala Lys Arg Pro Lys IleSer Phe Ser Asn Ile Ile Ser Asp Met 195 200 205 Lys Val Ala Arg Ser AlaThr Ala Arg Val Arg Ser Arg Pro Glu Leu 210 215 220 Arg Ile Gln Phe AspGlu Gly Tyr Asp Asn Tyr Pro Gly Gln Glu Lys 225 230 235 240 Thr Asp AspLeu Lys Lys Arg Lys Asn Ile Phe Thr Gly Lys Arg Leu 245 250 255 Asn IlePhe Asp Met Met Ala Val Thr Lys Glu Ala Pro Glu Thr Asp 260 265 270 ThrSer Pro Ser Leu Trp Asp Val Glu Phe Ala Lys Gln Leu Ala Thr 275 280 285Val Asn Glu Gln Pro Leu Gln Asn Gly Phe Glu Glu Leu Ile Gln Trp 290 295300 Thr Lys Glu Gly Lys Leu Trp Glu Phe Pro Ile Asn Asn Glu Ala Gly 305310 315 320 Phe Asp Asp Asp Gly Ser Glu Phe His Glu His Ile Phe Leu GluLys 325 330 335 His Leu Glu Ser Phe Pro Lys Gln Gly Pro Ile Arg His PheMet Glu 340 345 350 Leu Val Thr Cys Gly Leu Ser Lys Asn Pro Tyr Leu SerVal Lys Gln 355 360 365 Lys Val Glu His Ile Glu Trp Phe Arg Asn Tyr PheAsn Glu Lys Lys 370 375 380 Asp Ile Leu Lys Glu Ser Asn Ile Gln Phe Asn385 390 395 1314 base pairs nucleic acid single linear not providedHUVENOB01 35842 2 GACGTGTTTG GCAGCGGGAC GCACCATTTC AGTTGTGTTC TTGGTTCATTTCGTGTCTCG 60 GCGATGTTTC CTAGAGTCTC GACGTTCCTA CCTCTTCGCC CCCTTTCCCGCCACCCTTTG 120 TCCTCTGGAA GCCCGGAGAC ATCAGCGGCT GCGATTATGC TACTCACTGTTCGGCACGGA 180 ACAGTCAGGT ACCGCAGTTC AGCGCTGTTG GCCCGGACAA AAAATAACATCCAAAGATAT 240 TTTGGCACTA ACAGTGTGAT CTGTAGCAAG AAAGATAAGC AGTCTGTTCGAACTGAGGAG 300 ACTTCCAAGG AGACTTCAGA GAGCCAAGAC AGTGAAAAGG AAAATACGAAAAAAGACTTG 360 TTAGGCATTA TTAAGGGCAT GAAAGTTGAA TTAAGCACAG TAAATGTACGAACAACAAAG 420 CCCCCCAAAA GAAGACCACT TAAAAGTTTG GAAGCTACAC TTGGCAGGCTTCGAAGAGCT 480 ACAGAATATG CTCCAAAGAA GAGAATTGAG CCCCTGAGTC CTGAGTTGGTGGCAGCTGCA 540 TCTGCTGTGG CAGATTCTCT CCCTTTTGAT AAGCAAACAA CCAAGTCAGAGCTGCTGAGC 600 CAGCTCCAGC AGCATGAGGA AGAGTCAAGG GCACAGAGAG ATGCAAAGCGACCTAAAATT 660 AGTTTCAGTA ACATAATATC AGATATGAAA GTTGCCAGAT CTGCTACAGCTAGAGTTCGT 720 TCAAGACCAG AGCTTCGGAT TCAGTTTGAT GAAGGCTATG ACAATTATCCTGGCCAGGAG 780 AAGACGGATG ATCTTAAAAA AAGGAAAAAT ATATTCACAG GGAAAAGACTTAATATTTTT 840 GACATGATGG CAGTTACTAA AGAAGCACCT GAAACAGACA CATCACCTTCACTTTGGGAT 900 GTGGAATTTG CTAAGCAGTT AGCCACAGTA AATGAACAAC CCCTTCAGAATGGATTTGAA 960 GAGCTGATCC AGTGGACAAA AGAGGGGAAA CTATGGGAGT TCCCAATTAACAATGAAGCA 1020 GGTTTTGATG ATGATGGTTC AGAATTTCAT GAACATATAT TTCTGGAGAAACACCTGGAG 1080 AGCTTTCCAA AACAAGGACC AATTCGCCAC TTCATGGAGC TGGTGACTTGTGGCCTTTCC 1140 AAAAACCCAT ATCTTAGTGT TAAACAGAAG GTTGAACACA TAGAGTGGTTTAGAAATTAT 1200 TTTAATGAAA AAAAGGATAT TCTAAAAGAA AGTAACATAC AGTTCAATTAAGACCATGGA 1260 AATTTTTATT TCAAACAATT AGAGATGGAT ATTACAACTA AATAAAATAATTGC 1314 384 amino acids amino acid single linear not provided GenBank1272669 3 Met Leu His Arg Ile Pro Ala Phe Leu Arg Pro Arg Pro Phe SerGly 1 5 10 15 Leu Pro Leu Ser Cys Gly Asn Arg Asp Val Ser Val Ala ValLeu Pro 20 25 30 Ala Ala Gln Ser Gly Ala Val Arg Thr Glu Asn Asn Ile GlnArg His 35 40 45 Phe Cys Thr Ser Arg Ser Ile Cys Ser Lys Lys Val Asp GlnSer Val 50 55 60 Pro Ala Asn Glu Ile Ser Gln Lys Ala Ala Glu Ser Gln GlyArg Gly 65 70 75 80 Lys Glu Thr Leu Lys Lys Asp Leu Leu Asp Ile Ile LysAsp Met Lys 85 90 95 Val Asp Leu Ser Thr Ala Asn Val Lys Thr Pro Lys ProArg Gly Arg 100 105 110 Lys Pro Ser Ala Ser Leu Glu Ala Thr Val Asp ArgLeu Gln Lys Ala 115 120 125 Pro Glu Asp Pro Pro Lys Lys Arg Asn Glu PheLeu Ser Pro Glu Leu 130 135 140 Val Ala Ala Ala Ser Ala Val Ala Asp SerLeu Pro Phe Asp Lys Gln 145 150 155 160 Thr Thr Lys Ser Glu Leu Leu ArgGln Leu Gln Gln His Glu Glu Glu 165 170 175 Leu Arg Ala Gln Lys Asp ArgGlu Lys Arg Arg Ile Ser Phe Thr His 180 185 190 Ile Ile Ser Asn Met LysIle Ala Lys Ser Pro Ser Gly Arg Ala Ser 195 200 205 Thr Arg Pro Gln HisGln Ile Gln Phe Asp Glu Asp Met Asp Ser Ser 210 215 220 Leu Lys Gln GluLys Pro Thr Asp Phe Arg Lys Arg Lys Tyr Leu Phe 225 230 235 240 Lys GlyLys Arg Leu Ser Ile Phe Ala Asp Lys Ala Phe Ala Asp Glu 245 250 255 ProPro Glu Pro Glu Ala Ser Pro Ser Leu Trp Glu Ile Glu Phe Ala 260 265 270Lys Gln Leu Ala Ser Val Ala Asp Gln Pro Phe Glu Asn Gly Phe Glu 275 280285 Glu Met Ile Gln Trp Thr Lys Glu Gly Lys Leu Trp Glu Phe Pro Val 290295 300 Asn Asn Glu Ala Gly Leu Asp Asp Asp Gly Ser Glu Phe His Glu His305 310 315 320 Ile Phe Leu Asp Lys Tyr Leu Glu Asp Phe Pro Lys Gln GlyPro Ile 325 330 335 Arg Leu Phe Met Glu Leu Val Thr Cys Gly Leu Ser LysAsn Pro Tyr 340 345 350 Leu Ser Val Lys Gln Lys Val Glu His Ile Glu TrpPhe Arg Asn Tyr 355 360 365 Phe Asn Glu Lys Arg Asp Ile Leu Lys Glu AsnAsn Ile Ala Phe Thr 370 375 380

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence of SEQ ID NO: 1
 2. A method for producing a polypeptide ofclaim 1 , the method comprising: a) culturing a cell under conditionssuitable for expression of the polypeptide, wherein said cell istransformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1 , and b) recoveringthe polypeptide so expressed.
 3. A method for detecting a transcriptencoding a polypeptide in a sample, the method comprising: a)hybridizing a polynucleotide which encodes the polypeptide of claim 1with the sample containing nucleic acids, b) detecting complex formationbetween the polynucleotide and at least one nucleic acid of the sample,wherein complex formation indicates the presence of the transcript ofthe polypeptide in the sample.
 4. The method of claim 3 , wherein thenucleic acids of the sample are amplified prior to hybridization.
 5. Acomposition comprising an effective amount of a polypeptide of claim 1and an acceptable excipient.
 6. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1 , the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample. if present,the amount thereof.
 13. A method for detecting a target polynucleotidein a sample, said target polynucleotide having a sequence of apolynucleotide of claim 10 , the method comprising: a) amplifying saidtarget polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 14. A method for screening a compound foreffectiveness in altering expression of a target polynucleotide, whereinsaid target polynucleotide comprises a polynucleotide sequence of claim10 , the method comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 15. A method for assessing toxicityof a test compound, said method comprising: a) treating a biologicalsample containing nucleic acids with the test compound; b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 10 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 10 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 16. Apurified antibody which specifically binds to the polypeptide of claim
 1. 17. The antibody of claim 16 , wherein the antibody is: (a) a chimericantibody; (b) a single chain antibody; (c) a Fab fragment; (d) a F(ab′)₂fragment; (e) a Fv fragment; or (f) a humanized antibody.
 18. Apharmaceutical composition comprising an antibody of claim 16 and apharmaceutically acceptable excipient.
 19. A method of diagnosing acondition or disease associated with the expression of AUTOP in asubject, comprising administering to said subject an effective amount ofthe pharmaceutical composition of claim 18 .
 20. A pharmaceuticalcomposition of claim 18 , wherein the antibody is labeled.
 21. A methodof diagnosing a condition or disease associated with the expression ofAUTOP in a subject, comprising administering to said subject aneffective amount of the pharmaceutical composition of claim 20 .
 22. Amethod of preparing a polyclonal antibody with the specificity of theantibody of claim 16 comprising: a) immunizing an animal with apolypeptide of SEQ ID NO: 1 or an antigenically-effective fragmentthereof under conditions to elicit an antibody response; b) isolatinganimal antibodies; and c) screening the isolated antibodies with thepolypeptide thereby identifying a polyclonal antibody binds specificallyto a polypeptide of SEQ ID NO:
 1. 23. An antibody produced by a methodof claim 22 .
 24. A pharmaceutical composition comprising the antibodyof claim 23 in conjunction with a suitable pharmaceutical carrier.
 25. Amethod of making a monoclonal antibody with the specificity of theantibody of claim 16 comprising: a) immunizing an animal with apolypeptide of SEQ ID NO: 1 or an antigenically-effective fragmentthereof under conditions to elicit an antibody response; b) isolatingantibody producing cells from the animal; c) fusing the antibodyproducing cells with immortalized cells in culture to form monoclonalantibody-producing hybridoma cells; d) culturing the hybridoma cells;and e) isolating from the culture monoclonal antibodies which bindsspecifically to a polypeptide of SEQ ID NO:
 1. 26. A monoclonal antibodyproduced by a method of claim 25 .
 27. A pharmaceutical compositioncomprising the antibody of claim 26 in conjunction with a suitablepharmaceutical carrier.
 28. The antibody of claim 16 , wherein theantibody is produced by screening a Fab expression library.
 29. Theantibody of claim 16 , wherein the antibody is produced by screening arecombinant immunoglobulin library.
 30. A method for detecting apolypeptide of SEQ ID NO: 1 in a sample comprising the steps of: a)combining the antibody of claim 16 with a sample under conditions toallow specific binding; and b) detecting specific binding, whereinspecific binding indicates the presence of polypeptide of SEQ ID NO: 1in the sample.
 31. A method of using an antibody to purify polypeptideof SEQ ID NO: 1 from a sample, the method comprising: a) combining theantibody of claim 16 with a sample under conditions to allow specificbinding; and b) separating the antibody from the protein, therebyobtaining purified polypeptide of SEQ ID NO: 1.